FR2568682A1 - METHOD FOR DIAGNOSING HUMAN PAPILLOMAVIRUS INFECTIONS, MONOCLONAL ANTIBODIES AGAINST A TYPE OF HUMAN PAPILLOMAVIRUSES AND HYBRIDOMIC LINES HAVING THEM - Google Patents

Abstract

THE PRESENT INVENTION CONCERNS A PROCESS FOR DIAGNOSING HUMAN PAPILLOMAVIRUS, MONOCLONAL ANTIBODIES DIRECTED AGAINST A TYPE OF HUMAN PAPILLOMAVIRUS AND THE HYBRID CELL LINES WHICH SECRET THEM. THESE HYBRIDOMAS RESULT FROM THE FUSION OF MURINE MYELOMA CELLS AND MICE RATE CELLS THAT HAVE BEEN IMMUNIZED AGAINST THIS VIRUS. THESE ANTIBODIES ARE USEFUL FOR THE SPECIFIC IDENTIFICATION, ASSESSMENT AND PURIFICATION OF HUMAN PAPILLOMAVIRUS.

Description

The present invention relates to a dia-

  diagnosis of human papillomavirus infections using monoclonal antibodies to distinguish

  benign papillomavirus of potential papillomavirus-

  oncogenes. Classical methods of immunological reaction

  antigen / antibodies can not be systematically used

  to detect the presence of certain types of papillo-

  human mavirus because of the small amount available

  potentially oncogenic papillomavirus

  immunization of animals for the preparation of immuno-

  polyclonal sera.-It has therefore appeared necessary to

  at the point of a differential identification process

  distinguishing between human papillomaviruses (HPV), type 1 for example, papillomavirus type 16 or 18 whose oncogenic potential is known (refer to the article by Lionel Crawford, Nature, 1984, volume 310 p.l6) . To carry out the process according to the invention, monoclonal antibodies are prepared, starting from

  hybridoma lines specific for each type of papilloma

  human mavirus. As an example of implementation of the invention, monoclonal antibodies directed against human papillomavirus type 1 and cell lines

  hybrids that secrete these antibodies, have been prepared.

  The present invention relates to a method of

  diagnosis of human papillomavirus (HPV) using

  monoclonal antibodies directed against a type of human papillomavirus, hybrid cell lines that secrete these antibodies and the application of these antibodies to specific identification, assay and purification

papillomaviruses.

  Papillomaviruses of the papovavirus family cause, in humans, benign skin and mucosal epithelial tumors, warts and condylomas, which can sometimes degenerate into malignant tumors. So far, twenty-eight types of human papillomavirus have been counted, taking into account cross-hybridization rates.

  DNAs less than 50%; recently the appearance of can-

  genital and cutaneous has been associated with the presence of certain types of these viruses, type 1 among others, being considered perfectly benign.

  If it has been possible to show the existence of specific antigens

  of the different types, and antigens of the papillomavirus group, polyclonal antibodies specific for most of these types of viruses have never been prepared.

  because of the very small amount of antigenic material

  that available. It is however particularly desirable to have antibodies that are specific, that is to say, recognizing only one type of papillomavirus, to prepare reagents for identifying, differentiating, dosing and purifying these viruses, to the extent that some

  only are associated with carcinogenesis phenomena.

  In addition, these antibodies would allow the study of

antigenicity of these viruses.

  Thus, the development of a reagent for differentiating a benign virus, such as type 1, from potentially oncogenic viruses, such as those of type 16 or 18, was particularly desirable, and this is one of the fundamental advantages of the invention to allow this differentiation.

  Currently, there is no way of detecting

  of the types of papillomavirus, based on the reaction

  antigen / antibody, the benefits of which are known.

  We know, since the work of Kohler and Milstein

  published in 1975 in Nature 256 p.495, to make hybrids

  domes, immortal cell lines, by fusion of mouse myeloma cells and mouse spleen cells. The latter having been previously immunized against a given antigen, the hybridomas will secrete antibodies directed against this antigen and these antibodies will be monoclonal because of the selection technique and culture used. Although this method has been widely applied since then, it is not obvious to select the cell line that will secrete

  specific antibodies against a given type of papillo-

  mavirus, that is to say will not give or little reaction

crossed with other types.

  The hybridomas which are the subject of the invention are secretants of the antibodies specific for a particular type of human papillomavirus and these monoclonal antibodies are another subject of the invention. The human papillomavirus

  type 1 (or HPVl) is particularly suitable for

  of viruses infecting humans. Significant amounts of these viral particles are found in deep plantar warts. Monoclonal antibodies directed against the type 1 virus and the hybridomas which

  secrete, illustrate the present invention.

  According to the invention, the hybridomas are prepared by fusion of appropriately selected mouse myeloma cells and cells extracted from the spleen of mice previously immunized against the papillomavirus of the chosen type. In the case of type 1 papillomavirus, virus particles are extracted from human deep plantar warts, purified for injection into mice. It can be ensured that this viral preparation comprises only virus-type 1 by isolating the DNA present in a sample and verifying that this DNA gives the fragments known to be those of the HPVI virus, by endonuclease action by example Hind II and Hind III. The viral preparation is used to immunize mice by subcutaneous or intraperitoneal injection. We will make several successive injections, at intervals of several weeks. When the mice are immunized, they are killed, and their spleens removed; the cells of these rats

are used for the merger.

  Different lines of murine myeloma cells can be used to effect the fusion, they must allow the subsequent selection of the hybridomas formed by methods known per se, cells not secreting immunoglobulins and the SP2 / 0 strain are preferred. 8-azaguanine-resistant is ideally suited for hybridoma separation from non-fused cells. Fusion is achieved by contacting the two kinds of cells in the presence of a fusion agent which may be polyethylene glycol or the Sendai virus. After fusion, all the cells after dilution are cultured in a selective medium, where only the hybridomas can develop; in the case of SP 2/0 myeloma strains, the presence in the culture medium of azaserine will cause the disappearance of spleen cells that have not fused. Only a few cells are cultured per well of a conventional plate and after a few days, from 5 to 15, when the development of the cells has become apparent, the presence is sought,

  in the supernatant of the different wells,

  HPVI by a classical method (immunofluorescence, immune

  For example, antibodies produced by their reaction with HPVI antigens present in frozen plantar warts, immunofluorescence-mediated or immunoenzymatic assay can be detected; by this method, specific antibodies will be detected

  of a type or group of papillomaviruses. By immunodiffu-

  The use of whole viral particles as antigens will only highlight the specific antigens of one type of virus. Monoclonal antibodies can also be detected by the "Elisa" method

  using viral particles intact as anti-

  Genoa. The cells secreting interesting antibodies, thus highlighted, are then cloned by the technique

  limiting dilution to obtain monoclonal cultures.

  The monoclonal antibodies secreted by these cell clones can be isolated in the supernatants of their cultures, or in the ascites liquids which are formed after intraperitoneal injection into mice, hybridomas.

  The identification of the class to which

  immunoglobulins secreted by the hybridomas can be carried out by immunodiffusion according to a method

a nt -

  known, using mouse antibodies to mouse immunoglobulins (anti-IgG1, anti-IgG2a,

anti-IgG2b, anti-IgG3).

  Two hybridoma lines are the subject of the invention.

  tion. They have been deposited in the Micro-

  Institut Pasteur organizations (France) on July 30th

  1984, under the numbers 1-324 and 1-325.

  Clone n 1-324 has the reference 334 B6, while clone n I-325 has the reference 405 DS. These hybridomas, which result from the fusion of SP 2/0 myeloma cells and Bal b / c mouse cells, have secreted for more than a year monoclonal antibodies directed specifically against the HPV1 virus; indeed, these diluted antibodies

  1/400 (v / v), to detect the presence of antigens

  HPV 1 in frozen plantar warts,

  while, diluted 1/10 (v / v), they give no reaction

  detectable by immunofluorescence or immuno

  enzymatic (revelation of peroxidase activity) in frozen sections of warts caused by papillomavirus

type 2, 3, 4, 5, 6 and 7.

  The monoclonal antibodies are secreted by the cultured cells in large amounts: the titers of the cultures which are the reverse of the highest dilution of supernatant revealing the infected nuclei in sections of frozen warts or giving a precipitation line with viral particles, are between 10 and 100 in the case of immunofluorescence or dimmu.Orxydasee technique between 1 and 16 for the technique

immunodiffusion.

  Ascites liquids resulting from intraperitoneal injection into Bal b / c mice of 2 × 10 6 hybridomas have a titer, 12 days after injection, ranging from 200 to 400 (immunofluorescence determination) or from 200 to 400 ( determination by immunoperoxidase technique) or from 50 to 200 (immunodiffusion determination). The classes to which these antibodies belong have been determined by immunodiffusion; these are the subclass

  IgG1 (strain 334 86) and the subclass IgG2a (strain 405 D5).

  In the case of human papillomaviruses of other types, to the extent that they are only available in small quantities, for example because we do not know how to cultivate them, we can prepare antibodies specific to

  sufficient quantity using hyperimmunization techniques.

  as for example that described by LANGE et al. in 3. of Immunological Methods (1983) vol. 63 p.123-131 and which consists in coupling the viral antigen to

a lipopolysaccharide.

  The monoclonal antibodies according to the invention, specifically

  of a type of virus, are advantageously used to prepare immunological reagents for the detection, identification, assay and purification of the corresponding type of virus by methods whose principle is

monoelonaux-

  known in itself. The application of these antibodies / üaprepara-

  These immunological reagents are another object of the invention. HPV1 identification reagents containing monoclonal antibodies specific for HPV1 will be useful in determining the severity of HPV1 infections.

  papillomavirus. Another object of the invention is a

  assigned to determine the presence or absence of a lesion of potentially oncogenic papillomaviruses. It consists in carrying out an immunological test, the principle of which is known, between viral particles or viral antigens of the lesion with the monoclonal antibodies according to the invention.

  type HPV1 or any other type of papillo-

human mavirus.

  In the following, we illustrate the processes used to

  to prepare hybridomas and monoclonal antibodies

clones, according to the invention.

EXAMPLE

  a) Immunization of animals. The viral HPV1 particles were isolated and purified according to the method described in 3. Virol 24

108-120 (1977).

  Subsequently, three Bal b / c mice were injected subcutaneously with a suspension of these particles in phosphate buffered saline, mixed with an equal volume.

  Freund's complete adjuvant; each injection corresponds to

  at 50 μg of viral proteins. The injection was repeated

  after 2 weeks; then one month after the first injection, the same amount of viral particles were injected intraperitoneally to the animals, without adjuvant; the mice were killed three days after the last

  injection and their spleens removed.

  b) Fusion The cells of the spleens, approximately 2 × 10 8 cells per animal after isolation and washing with RPMI 1640 medium supplemented with 20% fetal calf serum, were fused with SO 2 / O mouse myeloma cells, resistant to 8-azaguanine. Approximately one myeloma cell was contacted with 10 cells of

  spleen, in the presence of 1 ml of 50% polyethylene glycol

  1 minute 30 to 370C. The hybridomas were then separated from unfused cells by culturing on a conventional medium consisting of RPMI 1640, supplemented with 20% fetal calf serum, hypoxanthine (10M) and azaserine (5x105M). . The RPMI medium commonly used for cell cultures is marketed by

  GIBCO - P.O.Box 35 - Paisley - Scotland. We filed

  5 x 105 cells per culture well.

  c) Search for the secreting hybridomas The fused cells had been distributed in wells and 12 days after the start of the culture, it was determined whether the supernatants of each of these wells contained anti-HPV1 antibodies, by the methods previously mentioned in FIG. Virology (1977) 24 p.108-120

and Virology (1978) 91 p. 243-255.

  Two wells were found in which the supernatural

  giants gave a positive result by the method of immu-

  nofluorescence or immunoperoxidase (on freeze

  warts) and by the immunodiffusion method (against whole viral particles). These two wells

  therefore contained antibodies specific for HPV1.

  d) Cloning The corresponding crops called 86 and D5

  have been cloned by dilution according to a principle

  naked, so that the cultures in each well are derived from only one cell. Supernatants from these wells were studied when growth was

  sufficient to keep only the wells or

  monoclonal bodies were present. We thus isolated two

clones, 334 B6 and 405 DS.

  (e) Identification of viral polypeptides bearing the

  antigenic determinants recognized by monoclonal antibodies

  clones secreted by clones 334 B6 and 405 D5.

  Viral particles are known to dissolve

  at 100 C by the action of a detergent such as SDS (sodium dodecyl sulphate) (conc.3%), in the presence of 4M urea and 2-mercaptoethanol (final concentration 5%), in three kinds of separable polypeptides by electrophoresis in polyacrylamide gel: one in large quantity, with an apparent molecular mass of 54,000 daltons, a second, in a smaller quantity, with a mass of 76,000 daltons and others with masses of between 12,500 and 16,500 daltons, as

  described in the two articles mentioned above.

  The monoclonal antibodies secreted by the cells of line 334 B6 and 405 D5 react with the polypeptides of mass 76 000 daltons and mass 54 000 daltons which are therefore both carriers of antigenic determinants of the virus. This has been demonstrated by the

  immunoblotting technique (Western Blot).

9 y c o2568682o

Claims (9)

  1. Method for determining the presence of human papillomaviruses, characterized in that an immunoreaction search is carried out between the viral particles or viral antigens present in a biopsy section or extracts of a lesion, and monoclonal antibodies specific to papillomaviruses.   2.An monoclonal antibody, characterized in that   is specifically directed against one type of papillomavirus.   3. monoclonal antibody according to claim 2, characterized in that it is directed specifically against the human papillomavirus type 1   4. Monoclonal antibody according to one of the claims   2 or 3, characterized in that it is secreted by a hybridoma resulting from the fusion of murine myeloma cells and mouse spleen cells immunized against a type of human papillomavirus.   Monoclonal antibody according to claim 3,   characterized in that it is secreted by the cell line   hybrids deposited at the National Collection of Microorganism Cultures at the Institut Pasteur (France) under the n 1-324.   6. Monoclonal antibody according to claim 3, characterized in that it is secreted by the line of   Hybrid cells deposited at the National Collection of Cul-   microorganisms at the Institut Pasteur (France) under the n 1-325.   7. Hybrid cell line secreting antigens   body against human papillomavirus type I, deposited in the National Collection of Culture of Microorganisms in   Institut Pasteur (France) under number 1-324.   8. Hybrid cell line secreting antibodies against human papillomavirus type 1, deposited in the National Collection of Culture of Microorganisms at   Institut Pasteur (France) under the n 1-325.   9.Application of a specific monoclonal antibody   that according to any one of claims 2 to 6, to 2568682   differentiation, detection, dosing or purification   cation of virus particles or specific antigens   different types of human papillomaviruses.   Application of a specific monoclonal antibody   of type I papillomavirus according to one of the   cations 3 to 6 for differentiation, detection, dosing and   ge or purification of viral particles or   specific genes of different, * human apillomaviruses